ORIGINAL ARTICLE
Assay Development for Histone Methyltransferases
Kurumi Y. Horiuchi, Mia M. Eason, Joseph J. Ferry, Jamie L. Planck, Colin P. Walsh, Robert F. Smith, Konrad T. Howitz, and Haiching Ma
Department of Biochemistry, Reaction Biology Corp., Malvern, Pennsylvania.
ABSTRACT Epigenetic modifications play a crucial role in human diseases. Unlike
genetic mutations, however. they do not change the underlying DNA
sequences. Epigenetic phenomena have gained increased attention in
the field of cancer research, with many studies indicating that they are
significantly involved in tumor establishment and progression. Histone
methyltronsferases (HMTs) ore o Iorge group of enzymes that specifically
methylate protein lysine and arginine residues, especially in histones,
using S-adenosyl-t-methionine (SAM) as the methyl donor. However, in
general, HMTs have no widely accepted high-throughput screening
(HTS) assay format, and reference inhibitors are not available for many
of the enzymes. In this study, we describe the application of a minia
turized, radioisotope-based reaction system: the HotSpofM platform for
methyltransferases. Since this platform employs tritiated SAM as a
cofactor, it can be applied to the assay of any HMT. The key advantage
of this format is that any substrate can be used, including peptides,
proteins, or even nucleosomes, without the need for labeling or any
other modifications. Using this platform, we have determined substrate
specificities, characterized enzyme kinetics, performed compound pro
filing far both lysine and arginine methyltransferases, and carried out
HTS for a small-library LOPAC against DOTIL After hit confirmation
and profiling, we found that suramin inhibited DOTIL, NSD2, and PRMT4
with /C50 values at a low pM range.
INTRODUCTION
E pigcnctics involves the study of changes in the regulation of gene activity and expression, independent of gene sequences. Post-translational modifications of histones, including methylation, acetylation, phosphorylation, and
ubiquitination, arc all important epigenetic factors. Histoncs arc proteins around which DNA is wound for compaction and gene regulation. Two copies each of histones H2A, I-128, H3, and H4 assemble to form one nucleosome core, which is wrapped by -146 base pairs of DNA. Histone HI binds the nuclcosome at the entry and
exit sites of the DNA and locks it into place. 1 The tight nucleosome structure helps to pack the enti re genome into the cell nucleus and can restrict the access of nuclear factors to the DNA. This inherently restrictive environment must be tightly regulated to ensure that permissive cellular processes such as gene transcription, repl ication, recombination, and repair occur only under the appropriate circumstances. Because the dysfunction of these systems is inherent in many disease states, epigenetics has become an emerging frontier fo r drug discovery.
The human genome encodes more than 70 enzymes that catalyze the methylation of lysine (Lys, K) or arginine (Arg, R) residues on histones HI , H2A, H2B, H3, and 1-14. They are collectively referred to as histone methyltransferases (HMTs). HMTs mainly methylate histones using S-adenosyl-L-methionine (AdoMet or SAM) as a methyl donor.2.J Most histone lysine methyltransferascs (HKMTs) contain a SET -domain [Su(var)3-9, Enhancer ofZeste, and Trithorax]. The only known HKMTs that lack the SET domain are the members of the DOT! family.1
Irregular expression of HKMTs is associated with human cancers.5·6 Enhancer of zeste homolog 2 (EZH2) is ubiquitously expressed during early embryogenesis, and becomes restricted to the central and peripheral nervous systems and the sites of feta l hematopoiesis during later dcvelopment.7 EZH2 is responsible for the methylation of Lys 27 of histone H38 as well as that of Lys 26 on histone H 1.9 Ovcrexpression of EZH2 is found in many cancers: prostate (metastatic), breast, liver, bladder, colon, skin, and lung, among others. However, surprising findings by Ernst and colleagues 10 have suggested that EZI-12 is a tumor suppressor in myeloid malignancies. G9a and G9a-like protein (GLP), the two highly homologous HKMTs, methylate Lys 9 of histone H3 and have also recently been reported to inactivate p53 via methylation of Lys 373.11
G9a and GLP arc ovcrexpresscd in various cancers, 11 suggesting that they arc putative oncogenes and potential inhibitory targets for cancer treatment. Gaughan and colleagues 12 found that SET7/9 methylation of the androgen receptor at Lys 632 enhances transcriptional activation of target genes, that SET7/9 expression is uprcgulated in prostate cancer tissue, and that SET7 (9 is proproliferative and antiapoptotic in prostate cancer cells. The non-SET HKMT, hOOT! L, mcthylatcs Lys 79 in the globular region of histone H3 4 and is reported to be associated with leukemogenesis. 13
The protein arginine methyltransferases (PRMTs) compose a smaller group of enzymes than the HKMTs. Only I I isoforms have
ABBREVIATIONS: EZH2, enhancer of zeste homolog 2; FP, fluorescence polarization; GLP, G9a-like protein; 3H-SAM, S-adenosyi-L-[methyi-3H)methionine; HKMT, histone
lysine methyltransferasc; HMT, histone methyltransferase; HTS, high-throughput screening; ICso. half-maximal inhibitory concentration; MMA, monomethyl arginine;
PRMTs, protein arginine methyltransferascs; SAH, 5-adcnosyi-L-homocysteine; SAHH, 5-adenosylhomocystcine hydrolase; SAM, 5-adenosyi-L-methioninc; SET, Su(var)3-9,
enhancer of zcste, and trithorax; uHTS, ultrahigh-throughput screening.
001: 10.1089/adt.2012.480 © MARY ANN LIEBERT, INC. • VOL. XX NO. XX • XXXX 2013 ASSAY and Drug Development Technologies 1
HORIUCHI ET AL.
been discovered thus far: PRMT 1-PRMT I I. 14 All of these enzymes display conserved sequence motifs in the catalytic domain, although methyl transferase activities have not yet been confirmed for PRMTIO and PRMTII. The PRMT enzymes have been classified according to the nature of the dimcthylated arginine reaction product. The type-I PRMT enzymes catalyze the formation of monomcthyl arginine (MMA) and asymmetric dimethyl arginine, while the type n PRMT enzymes fo rm MMA and symmetric dimethyl arginine. The enzymes PRMT I, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8 belong to the type I group, whereas PRMT5, PRMT7, and PRMT9 arc type n enzymcs.1 5-18 Arginine methylation is an abundant post- translational modification that regulates a diverse array of cellular functions. The list of proteins known to be methylated on arginine has grown rapidly over the past decade and now includes hundreds of proteins. Recently identified PRMT substrates include nudcolin, fibrillarin, and hclicascs.17·19 Various biochemical and biological processes such as signal transduction, proliferation, transcriptional regulation, and RNA splicing are known to involve arginine methylation. In addition to the important role of PRMTs in normal cellular fu nction, dysregulatcd PRMT activities are implicated in disease processes such as certai n cancer types, cardiovascular disease, multiple sclerosis, and spinal muscular atrophy. 20
-22
Due to the role of HMTs as important epigenetic regulators and their dear link to human cancers, HMTs have generated intense interest as drug discovery targets. Although many HMT assay formats arc currently available, each has its own particular pros and cons (see the Discussion section), and there is no widely accepted highthroughput screening (HTS) assay format. In an effort to fill this gap, we have produced enzymes and applied a modified miniaturized radioisotope-based filter-binding assay for ITMTs. HotSpotsM. originally developed for kinases,21
"24 can be performed at the low costs
necessary to serve the drug discovery market. This platform detects total methylation of any substrate on both lysine and arginine residues. Substrates ranging from peptides to nuclcosomcs can be used without the need for modification; therefore, U1is platform is suitable for ultrahigh-throughput screening (uHTS) and selectivity profiling against a large panel ofHMTs. Here we will show the results ofHMT studies performed with this platform. The data indicate that this format enables HTS of HMTs as well as compound evaluations and ki netic studies.
MATERIALS AND METHODS Materials
Human DOTIL (residues 1- 4 16; accession II NM_032482) was expressed in Escllericllia coli as N-terminal GST fusions. Human recombinant EZH I (residues 2-747; Gcnbank Accession II
NM_OOI99 1) or EZH2 (residues 2- 746; Genbank Accession II
NM_OOI203247) were coexpressed with human recombinants AEBP2 (2- 5 17; NM_OOIII4176), EED (2-44 1; NM_003797), RbAp48 (2-425; NM_005610), and SUZ I2 (2- 739; NM_OI53 55) in an insect cell/baculovirus expression system to form the 5-member EZH I or EZI-12 complexes. All proteins were full length (residue 2 through Cterminus). The EED subunit incorporated an N-terminal Flag-tag, and
2 ASSAY and Drug Development Technologies xxxx 2013
all others included anN-terminal His-tag. Human GLP (residues 894-1298; accession II NM_024757) and Human G9a (residues 786- 121 0; accession II NM_006709) were expressed as N-terminal GST fusion protein in E. coli. Human MlL I (residues 3745-3969; accession II NM_005933), human WDR5 (22-334; NM_OI7588), RbBP5 (1-538; NM_005057), Ash2L (2-534; NM_00 1105214). and OPY-30 (1 -99; NM_0325742) were expressed in E. coli with N-tcrminal His-tags assembled as a complex and stored in 20 mM Tris-HCI, pH 7.5, 300 mM NaCI, I mM TCEP, 10% (w/v) glycerol, and I ~1M ZnCI 2•
Human MLL2 (residues 53 19-5537; accession # NM_003482), human MLLJ (residues 4689-49 11 ; accession # NM_ I70606), and human MLL4 (residues 2490-27 15; accession II NM_O 14727) were expressed in E. coli with N-terminal His-tags, and SET I B (residues 1629- 1923; accession II NM_O 15048) was expressed in E. coli with N-tcrminal GST-tag. All four were assembled in complexes as MLLI , as mentioned above. Human recombinant NSD2 (residues 2-1365; accession II NM_OO 1042424) was expressed with an N-tcrminal Histag in an insect cell/baculovirus expression system. Human recombinant SUV39H I (residues 44-412; accession II NM_003 173) and SUV39H2 (residues 48- 4 1 0; accession II NM_OO 1193424), both with C-terminal His-Tags, were expressed in E. coli. The following enzymes were codcvclopcd or purchased from BPS Bioscicnccs: E. coli
expressed SET7, His-tagged, full length; SETS, GST-taggcd aa l95-352; PRMTI, GST-tagged aa2-end; PRMT3, GST-taggcd aa2-cnd ; PRMT6, and His-tagged aa2-end, and Free Style"' 293-F cells (human kidney line; Invitrogen) expressed SETMAR, Flag-tagged aa 14-end; PRMT4, Flag-tagged aa2-cnd; and PRMT5, Flag-tagged aa2-end.
Nudcosomcs were prepared from Hela according to Schnitzler.25
Histone H3 protein was purchased from Sigma. Chicken core histoncs and human recombinant histone H4 were purchased from Millipore. Histone H3 peptide aa 1- 21 (ART KQT ARK STG GKA PRK QLA TKA A-NH2), histone H3 peptide aa 15- 39 (H-APR KQL ATK AAR KSA PAT GGV KKP H-ml), histone H4 peptide aa 1-2 1 (H-SGR GKG GKG LGK GGA KRH RKV-OH), and histone H3 peptide aa3- 17 (K9-
monomcthylated) were purchased from AnaSpec. [Histone pcptides arc henceforth referred to in the following format: H3 ( 1-2 1 ), H3 ( 15-39), etc.]
5-Adcnosyi-L- [mcthyi-3H]methionine eH-SAM) and streptavidincoatcd FlashPlatc were purchased from PerkinEimer. The control compounds 5-adenosyl-L-homocysteine (SAH), Sincfungin, chactocin, BIXO 1294, and suramin were purchased from Sigma. Suramin analogs, NF 110 and NF 449, were purchased from RElD Systems.
Methyltransferase Assay Methyltransferase assays were performed in the radioisotope
based llotSpot formal as described previously23•24 with the following
modifications. The reaction buffer for EZH I and EZH2 was 50 mM Tris-HCI, pH 8.0, 50 mM NaCI, I mM EDT A, I mM OTT, I mM PMSF, and 1% DMSO. The reaction buffer for all other HMTs was 50 mM Tris-HCI, pH 8.5, 50 mM NaCI, 5 mM MgCI2, I mM OTT, I mM PMSF, and I% DMSO. Standard substrate concentrations were 5 J.lM peptide or protein substrate, and I J.lM SAM, unless otherwise mentioned. For control compound IC50 determinations, the test compounds were
ASSAY DEVELOPMENT FOR HMTS
diluted in DMSO, and then added to the enzyme/substrate mixtures in
nanoliter amounts by using an acoustic technology (Echo550; Lab
cyte). The react ion was in itiated by the addition of 3 H-SAM, and
incubated at 30°( for I h. The reaction was detected by a filter
binding method. Data analysis was performed using Graph Pad Prism software for curve fits, and GraFit (Erithacus) for global lit of kinetic
studies using the ternary complex equation:
used in approaches that monitor SAM consumption and/o r SAH production. 26
Each lysine-speci fic HMT has a particular substrate specificity ;
each enzyme methylates specific residues on specific histones. For
example, DOT! L methylates Lys 79 of histone HJ ,' and G9a methylates Lys 9 of histone HJ and Lys 27 iu vitro,21 although methylation
on proteins other than histoncs has been reported. 11 PRMTs are able
to methylate arginine residues of a variety of protein substrates, in
cluding histones. To determine the proper substrates for each HMT
iu vitro, a collection ofi-IMTs were tested against all avail able histone
substrates. Table 1 summarizes the substrates that showed good ac
tivity fo r each enzyme. In ag reement with published data/ 8 DOTIL
did not methylate isolated histone 1-13 protein, and the activity was
only seen with nucleosomes or core histoncs. Some enzymes only
have activity wi th histone protein substrates or histone complexes,
( I)
where A is SAM; B is peptide substrate; K" and KB arc Michaelis
constants for each substrate; and K,' is the dissociation constant
for SAM.
Methyltransferase assay for G9a was also performed in a Flash
Plate. The reaction was performed under the same conditions as in the
HotSpot assay, but using biotinylatcd HJ ( 1-2 1)
(AnaSpec). After the termination of the reaction
by the addition of 0.1 mM SAH, the reaction was
transferred into the Flash Plate (Perkin Elmer) and
counted by TopCount (PerkinEimcr).
Table 1. Substrate Specificity of f-!istone Methyltransferases ~--:.·-~~- ··rt:t~rt~ .. ·~"-'··· · ~- -.~ I ~Vj.;.) 105/~,t_~;;t~;...;~~~-:· f-·J·-~.<~ ~ Reported methylation Enzyme Substrates showing good activity in HotSpot sites (from UniProtKB)
Compound Screening LOPAC (Sigma), a small library of 1,280 com
pounds, was screened against DOT! L with core
histone as the substrate in the assay described as
above. The compounds were tested in a single dose
at 20 IJM under the condition of 250 nM of DOT! L, 0.05 mg/ml of core histone, and 500 nM SAM
(dose to Km; see Supplementary Fig. S I for assay
optimization, available online at www.liebcrtpub
.com/adt) for 1-h reaction at 30°C. The assay pro
tocol is summarized in Supplementary Table S I.
The follow-up hit confirmation was performed
in a I 0-dose IC50 mode with a threefold serial
dilution starting at 100 J.IM under the same con
dition with the primary screening.
RESULTS Substra te Specificity
The HotSpot format measures total methyla
tion of a substrate by direct measurement of the
filter-bound tritiated substrate, without the need
for coupling enzymes or antibodies. Therefore,
any substrate can be used label- free. This rep
resents a major advantage over other detection
methods that require methylation-specific anti
bodies or peptide modifications such as biotin
labeled peptides in SPA assays and sequence
manipulation in the mobil ity-shift assay . In
addition, this radioisotope-based format is not
affected by compound fluorescence or by
the inhibition of coupling enzymes, the two
causes of false positives that arc commonly
DOTll
EZH1
EZH2
G9a
GLP
MLL1
MU1
Mll3
MLL4
NSD2
SET1B
SET7
SETS
SETMAR
SMY02
SUV39H1
SUV39H2
PRMT1
PRMT3
PRMT4
PRMTS
PRMT6
Nucteosomes H3K79
Nucteosomes, core histone, histone H3, H3 (21-44) H3K27
Nucleosomes, core histone, histone H3, H3 (21-44) H3K27
Core histone, histone H3, H3 (1-21) H3K9, H3K27, p53 K373
Core histone, histone H3, H3 (1-21) H3K9, H3K27, p53 K373
Nucleosomes, core histone, histone H3, H3 (1-21) H3K4
Nucleosomes, core histone, histone H3, H3 (1-21) H3K4
Nucleosomes, core histone, histone H3, H3 (1-21) H3K4
Nucleosomes, core histone, histone H3. H3 (1-21) H3K4
Nucleosomes H3K36, H4K20
Core histone H3K4
Core histone, histone H3, H3 (1-21) H3K4
Core histone, histone H4, H4 (1- 21) H4K20
Core histone, histone H3 H3K4, H3K36
Core histone, histone H3, histone H4, H3 (1-21}, H4 (1-21) H3K36
Core histone, histone H3, H3 (1 -21) H3K9
Core histone, histone H3, H3 (1-21) H3K9
Nucleosomes, core histone, histone H4, H4 (1-2 1) H4R3
Nucleosomes, core histone, histone H3, histone H4, H4 (1-21) Ribosomal protein
Core histone. histone H3, histone H4, H3 (1-21) H3R17
Core histone, histone H3, histone H4, H4 (1-21) H2A,H3R8. H4R3
Histone H3, histone H4, H4 (1-21) H3R2, H2A. H4R3
~ MARY ANN LIEBERT, INC. • VOL. XX NO. XX • XXXX 2013 ASSAY and Drug Development Technologies 3
HORIUCHI ET AL.
whereas others have activity with both the protein and peptide substrates. By using specifically methylated substrates, for example, dimethylated histone HJ (HJK9me2), the rates of particular methylation events (e.g., HJK9me2 to HJK9me3) can also be measured.
Kinetic Studies for HMTs Studies 10 determine kinetic constants, such as the K,.. values of
IIMTs, have been reported for several assay fo rmats. However, the values of kinetic constants may vary with the assay formats and conditions, and the reported kinetic constants of each enzyme from different assay formats can be hard to compare. The HotSpot formal is essentially similar to the traditional gold standard radioisotope detection used in conjunction with gel electrophoresis or mass spectroscopy for most reported mechanistic studies. Using radioisotopebased HotSpot, the K,.. was determined for several HMTs with different substrates. Reactions were performed as timecoursc measurements at varying concentrations of SAM and peptide/protein substrate.
The data demonstrate that methyl transferase activities were linear with time. Figure 1 shows the progress curves for the SUV39H2 methyltransfcrasc reaction, as an example, plotted against time at 5 ~1M of peptide substrate and varied concentrations of SAM. The reaction was linear up to 60 min at most SAM concentrations, or 90 min at I J.!M SAM. Similar experiments were performed at different concentrations of the peptide substrate with varying SAM concentrations. Taking the slope of the initial linear portion (signal/ timc=velocity), velocities were plotted against SAM concentrations (Fig. 2A) or peptide substrate concentrations (Fig. 28} to produce the Michaclis-Mcntcn plots. The Km values for SAM were not changed significantly by changing the peptide substrate concentrations (Fig.
2A). Similarly, the Km values for the peptide substrate did not change over any SAM concentrations tested (Fig. 28). To analyze further, the
60000
'0 50000 c:: j 0 ... 40000 Cl .X u
"' 30000 II? iii c:: 20000 Cl en
10000
0 0 25 50 75 100 125
Time (min)
Fig. 1. Progress curves for the SUV39H2 methyltransferase reac· tion. Reaction conditions are s J.JM of histone H3 (aa1-21) peptide substrate and varying concentrations of 5-adenosyl-L-methionine (SAM) at 0.125 J.!M (0), 0.25 J.!M (A), 0.5 J.!M (0), and 1 J.!M ( + ).
4 ASSAY and Drug Development Technologies xxxx 20 13
double reciprocal of Fig. 2A was plotted in Fig. 2C. As shown, lines converged at one point to the left of they-axis. This pattern excludes a possibili ty of double-displacement reactions (or Ping-Pong; first, the substrate/product must leave before second substrate binding), which would display parallel lines in double-reciprocal plots.29
Therefore, the SUV39fl2 methyltransferase reaction must be a random-ordered or compulsory-ordered Bi-Bi reaction, which means that SUV39H2 fo rms a ternary complex of the SUV39H2/SAM/ peptide substrate. At this point, it is difficult to distinguish between the two mechanisms: random ordered or compulsory ordered. To obtain accurate constants, a global-fit analysis was performed using GraFit software with the ternary complex equation (Eq. 1;
Fig. 2D). Global fits weigh equally on all data points; therefore, it is more reliable than the traditional graphical method, which is highly affected by imprecise low-activity data points. The global fits revealed that the K,.. value of SUV39H2 for SAM was 1.27 J.!M, and that for the peptide was 9.9 J.!M (Fig. 2D). Further studies with protein or methylated substrates arc needed to understand the mechanism of the SUV39H2 reaction in more detail, to determine, for example, whether the reaction is processive or not (i.e.,
different substrates with protein and/or methylated substrate, processive or not).
The kinetic constants for other HMTs were determined by similar experiments. Most methyltransfcrase reactions were linear with time up to 60 min, and others for even longer up to 90- 120 min. Taking slopes of the in itial linear portion of the reaction progress curves, the Michaelis-Menten plots were obtained for other HMTs with their corresponding substra tes. The obtained K,.. values by global fit arc summarized in Table 2. The K,.. value of SET7 for SAM was lower0
•31 or higher2 than that reported previously, depending
on the assay fo rmat. The SAM K,.. values for all lfMTs tested were lower than 5 J.!M, mostly lower than I J.!M under the conditions tested. Similarly, with a couple of exceptions, the K'" values for peptide or protein substrates were lower than I J.!M (Table 2). The kinetic constants of alternative substrates with the listed HMTs as well as the kinetic characterization of additional HMTs were determined. In general, the K,.. values for the protein substrate were low and difficult to obtain accurately. Thus, the SAM K'" values were obtained at a fixed substrate concentration (Table 3). The SAM K'" values at fixed substrate concentrations fall in a similar range to those in Table 2,
and are in generally a good agreement with reported values obtained by radioisotope-based assays.32
Compound Evalua tion The known mcthyltransferase inhibitors were tested in this assay
format against selected HMTs: G9a, SET7, and PRMT5. The compounds tested were SAil (5-Adcnosyi-L-homocysteine or AdoHcy), sincfungin, chactocin, and BIXO 1294. SAH is a product of the methyltransferase reaction, which inhibits HMTs competitively with respect to SAM. Sincfungin, a fungal compound, is an analog of cofactor SAM, chaetocin, an inhibitor of SUV39, and BIXOI294, an inhibitorofG9a/GLP.33-3~ As shown in Fig. 3, the IC~0 values for G9a, SET7, and PRMT5 with peptide and protein substrates arc obtained
ASSAY DEVELOPMENT FOR HMTS
A Michaelis-Menten Plot for SW39H2
8 M ichaelis-Menten Plot for Fig. 2 . Kinetic analysis of SUV39H2 with a peptide s ubstrate. The initial velocity of the SUV39H2 reaction was plotted as the MichaelisMenten plots for SAM {A) and for histone H3 (aa1-21) peptide sub· strate (B) using GraphPad Prism software. Peptide concentrations and Km values obtained by individual nonlinear regression in {A) are 2.5 and 1-46 (•). 5 and 2.38 (6), 10 and 2.oo (e), and 20 and 1.67 1-1M (0), respectively. SAM concentrations and obtained Km values in (B) are 0.125 and 5-77 c•>. 0.25 and 13.6 (6), 0.5 and 8.14 (e), 1 and 14.53 (0). 2.5 and 23.6 ( 0 ), 5 and 10.1 (0), and 10 and 10.91-1M (x ), respectively. Data from (A) were replotted as a double-reciprocal plot (C: symbols are corresponding to A), and global-fit (D) using Grafit software with the ternary complex equation (Eq. 1). Peptide concentrations in (D) are 2.5 (0), 5 (e), 10 (0), and 201-1M (.). and Km for SAM is 1.271JM, and Km for peptide substrate is 9.90 1JM, and the dissociation constant for SAM, KA' · is 2.58 1-1M.
c e 3000
=iij 2000 c: Cl
§. ~ 1000 :s ~
c
2.5 5.0 7.5 10.0
SAM (~M)
3000
c e :::: "' 2000 c: Cl
§. 1:- 1000 ·c:; 0 "i >
D 2500
2000
~ 1500 g a; > 1000
500
0 0 2
using the HotSpot rad ioisotope-based assay under the standard conditions. SAH and sinefungin each differed in thei r inhibition, although both compounds were SAM competitive: IC50 values of I.41JM and I 0 11M for G9a, 290 11M and 2.41JM for SET7, and 1.21JM and 306 nM for PRMT5, respectively, with a peptide substrate.
Table 2. Kinetic Constants for Histone Methyltransferases: Constants Obtained from Global Fit
Enzyme I Substrate I SAM Km (taM) I Substrate Km (jaM)
DOTll Core Histone 0.38±0.053 0.061±0.012 lmg/ml)
EZH2 Core Histone 0.42±0.072 0.012±0.005(mg/ml)
G9a H3(1-21) peptide 0.53±0.043 0.6±0.096 I
SET7 H3(1-21) peptide 0.22±0.074 5.7±0.031 I
SW39H1 Histone H3 0.56±0.014 0.53±0.042 I
SW39H2 H3(1-21) peptide 1.27±0.56 9.9±0.92 I
PRMT1 H4(1-21) peptide 0.28±0.047 0.24±0.089 I
PRMT3 H4(1-21) peptide 2.1 ±0.67 0.54+0.086 I
PRMT4 H3(1-21) peptide 3.1±0.46 0.32±0.069 I PRMT5 H4(1-21) peptide 1.07±0.21 0.11±0.075 I
SAM, 5-adenosyi-L-methionine.
5
SUV39H2
10 15 20
H3(1·21) (~M)
4 6 8 10
SAM ()1M)
Chaetocin inhibited both G9a and SET7 weakly with a peptide substrate IIC50 values of I81JM and 540 11M, respectively), but did not inhibit PRMT5. Interesti ngly, the G9a inhibitor BIX01294 also weakly inhibited SET7 and PRMT5 with a peptide substrate, but did not inhibit with a protein substrate. The IC50 value of BIXO 1294 for G9a with a peptide substrate was 5.31JM (Fig. JA), slightly higher than that reported.35 To va lidate the G9a assay with a peptide substrate in HotSpot, the G9a assay was also performed in the FlashPlate fo rmat with 0.51JM of biotinylated H3 ( 1-2 1) peptide at I 11M SAM. The 1( 50 value of BIXO 1294 was obtained with 5.0 11M, comparable with the value obtained with HotSpot (Fig. JA).
When the histone H3 protein was used as a substrate, the IC50
values were shifted up for most compounds, especially for BIXO 1294 (Fig. 38, D, F). These results suggest that the Km value fo r the histone protein was lower than that of the peptide substrate, thus making it more difficult for BIX01294 to displace the histone protein.
Since BIXO 1294 has been reported as a peptide substratecompetitive and SAM-uncompetitive inhibitor,n lower peptide (0.5 1JM) and higher SAM ( 10 11M) concentrations were tested (Fig. 4A).
Under this condition, the IC50 value was shifted to a lower value, 2.2 1JM, which is similar to that obtained by a mass spectrometry-based assay.34 On the other hand, the IC50 value ofSAH was shifted 10-fold higher when the SAM concentration was increased to I 0 11M (Fig. 4A). This shift was expected for a SAM competitive inhibitor. G9a can mono- and dimethylate the lysine 9 residue on histone H3. Therefore, dimethylation was monitored using monomethylated lysine 9 peptide as a substrate. As shown in Fig. 48, the IC50 value of BIXO 1294 was
1C> MARY ANN LIEBERT, INC. • VOL. XX NO. XX • XXXX 2013 ASSAY and Drug Development Technologies 5
HORIUCHI ET AL.
Table 3. Kinetic Constants for Histone Methyltransferases: 5-Adenosyl-t-Methionine Km at a Fixed Substrate Concentration
Enzyme I Substrate I SAM Km {JtM)
EZH1 Histone H3 1.24±0.15
EZH2 Histone H3 1.64±0.26
G9a Histone H3 0.74±0.10
GLP Core histone 0.95±0.18
GLP Histone H3 0.29±0.066
MLL1 Core histone 0.66±0.14
MLL1 Histone H3 0.50±0.067
MLL2 Core histone 4.50±0.82
MLL2 Histone H3 3.17±0.37
Mll3 Core histone 0.85±0.19
MLL3 Histone H3 0.96±0.18
sm Histone H3 1.64±0.12
SETS Histone H4 16.3±5.83
SET MAR Histone H3 1.13 ±0.42
SMYD2 Histone H3 0.12 ±0.013
SUV39H1 Histone H3 0.75±0.11
SUV39H2 Histone H3 0.74±0.23
PRMTl Histone H4 5.20±0.91
PRMT3 Histone H3 2.80±0.61
PRMT4 Histone H3 0.21 ±0.052
PRMTS Histone H3 0.70±0.17
PRMT6 Histone H3 2.20±0.47
Kinetic constants were determined at a concentration of SliM for histone or 0.05 mg/ml for core histone.
shifted about fivefold lower to I .311M compared to that with non
methy lated substnte (Fig. JA), a lthough the activity was < 1/ 10. Tri
methylation was minimal when using dimethylated lysine 9 peptide as
a substrate under this condition. On the other hand, the IC50 value of
SAH was not changed significantly (Figs. JA and 4B).
Overall, the data indicate that the HotSpot format is ideal for com
pound profiling, since any substrate can be used without any modifi
cations. In addition, the IC50 values obtained with this format are
similar to those from mass spectrometry-based assays, which are the
most reliable methods for detecting the methyl transferase reactions.
6 ASSAY and Drug Development Technologies xxxx 2013
Compound Screening Low-cost and robust assays with minima l false positives have been
desired for I-ITS, especially for difficult or expensive enzymes. DOT! L
is an attractive drug ta rget; however, it needs a histone octamer or nucJeosomcs as a substrate for enzyme activity, and monomer his
tone or peptide substrates do not work. Thus, screening against
DOTIL is costly and difficuli to run with certain assay fo rmats. We
screened LOPAC (Sigma), a small library of I ,280 compounds, against
DOTIL with core histone as the substrate in a miniaturized gold
standard radioisotope- based assay where the cost of the radioisotope
labeled cofactor and its waste, as well as that of the other reagents,
could be minimized. Althoug h most ~fMT assays in this fo rmat
have Z'- factors3G of >0.6, the Z'-factor was 0.52 fo r the DOT! L assay,
and the Signal:Background ratio was 4.3 (Supplementary Table 52).
We identified three compounds that showed more than 70% inhibi
tion against DOT! L-two of these were suramin and its analog, while
the other was L-cysteine sulfin ic acid.
The follow- up hi t confi rmation was performed by cherry-picking
o f three compounds under the same condition of primary screen ing.
Then, suramin and its 2 analogs, NF 110 and NF 449, were profiled
against 17 HMTs in our panel. The obtained IC50 values a re sum
marized in Table 4. Suramin and NF 449 showed similar inhibition
patterns, whereas NF 110 showed little activity. In teresti ngly, ac
tivities of many HMTs were increased by suram in and NF 449 at low
concentTations of compounds, while inhibited a t h igher concentra
tions. The degree o f such activa tions was not consistent; thus, the IC50
values may not be accu rate; these were expressed in italic letters. The
reason for such activations is not clear, whether there was real en
zyme activation, or were false signals caused by compound aggregation, etc. Since su ramin is a relatively large molecule, it could
possibly have caused compound aggregation involving substrates
and/or enzymes. Methyltransferases that were consistently inhibited
by suramin (wi thout apparent activation) are DOTIL, NSD2, and
PRMT4 with IC50 values at a low 11M range.
DISCUSSION The radioisotope-based miniaturized filter-binding assay, HotSpot
platform, was initially developed for kinase assays, but has since been
successfully applied to other transferase enzyme classes to serve
markets for ui-ITS, large-scale IC50 determinations, and selectivity/
toxicity profiling in drug discovery. 23·24 In this study, we have
modified this platform fo r ~fMT assays using tritium-labeled SAM as
a cofactor, and demonstrated that compound p rofiling, kinetic
studies, and I-ITS can be performed cost effectively with this system.
The currently available assay formats for HMTs have various
li mitatio ns. zG The traditiona l methods a re gel-based radioisotope
assays or mass spectrometry-based assays,37 wh ich directly measure
methylation on the substrate. Mass spectrometry-based assays are the
most reliable assays, and therefore detailed kinetic studies still em
ploy these formats. However, apply ing high-throughput formats with
these assays is difficult and/or requires expensive instrumentation.
Another radioisotope-based assay, first reported by Rathert et a1.,36
ASSAY DEVELOPMENT FOR HMTS
used streptavidin-coated FlashPlates to capture the biotinylated
peptide substrate, which, while bound to the plate, was enzymatically labeled with tri tiated SAM. This is a continuous assay and may be
applied to HTS. Aside from the requirement fo r biotinylated peptides, it is ha rd to control the substrate concentrations in this sys
tem, making the determination of ki netic parameters problematic.
Most popular assays are antibody based and employ a variety of detection systems, includi ng biotin-avidin, CLOT, and ELISA. The
CLOT assay is a homogeneous assay in which the methylation of
a biotinylated histone peptide is measured through methylationspecific antibody-based detection, in conjunction with strcptavidin
coated do nor and secondary antibody-coated AlphaScreen acceptor
A Compound ICSO for G9a/H3(1-21)
~ 80 > ti <
60
i!- 40
20
0 ·9 -8 ·1 -6 ·5 -4 ·3
Log [Compound) (M)
c Compound ICSO for SET7/H3(1·21)
120
100
~ 80 z u <
60
~ 0 40
20
0 ·9 -8 ·1 -6 · 5 -4 ..J
Log [Compound) (M)
E Compound ICSO for PRMT5/H4(1-21)
120
100
~ 80 ~ u <
60
~ 0 40
20
0 ..g -8 ·1 -6 ·5 -4 ..J
Log [Compound] (M)
8 Compound ICSO for G9a/H3
120
100
~ 80 > ti 60 < ~ 0 40
20
-8 ·7 -6 ·5 -4 ..J
Log [Compound] (M)
D Compound ICSO for SET7/H3
120
100
~ 80 ·:; ti 60 < ~ 40
20
0 .g -8 ·7 -6 .s -4 ..J
Log [Compound] (M)
F Compound IC50 for PRMT5/H4
20
o+---~--~~--~~~--~ ·9 -8 ·7 -6 · 5 -4 ·3
Log [Compound] (M)
Fig. 3· The JC50 determinations of control compounds for G9a , SET7, and PRMT5 at 1 ~M SAM with peptide or protein subs trates; (A) G9a and (C) SET7 with 5 ~1M of histone H3 (aa1-21) pe ptide as subs trate, (E) PRMT5 with 5 ~M of his tone H4 (aa1-21) peptide as subs tra te, (B) G9a and SET7 (D) with 5 ~M his tone H3 as subs trate, (F) and PRMT5 with 5 ~M of histone H4. Compounds tested are SAH (e), sinefungin ('i7 ), BIX0129 4 ( + ), and chaetocin (0), and the obtained IC50 values are (A) 1.45, 10.4, 5·3· and 17.8 ~M. respectively, (B) 7.1, 150, 320 , and 0.16 ~M. respectively, (C) 29 0, 2.38, 89.1, and 540 ~M. respectively, (D) 6o ~M fo r SAH and 9 .1 ~M for sinefung in, (E) 1.2 ~M for SAH, 0.31 ~M for sinefungin , and 140 ~M fo r BIX01294, a nd (F) 1.0 ~M for SAH and o.69 ~M for sinefungin .
beads.39 The limitation of such an
tibody-based detection systems is
the need for antibodies speci fic for
particular methylation sites as well
as fo r mono-, d i-, or trimethylation.
Therefore, it is di fficul t to profile a
compound against a wide selection
of HMTs with different substrates{
methylation sites or to perfo rm
kinetic studies. While the above
methods detect methylation of
(mostly) peptide substrates, another approach is to detect AdoHcy (5-
adenosyl- homocystcine, or SAH),
the reaction product derived from
AdoMet (SAM). Graves ct a /.40 have
described a competitive fluores
cence polarization (FP) assay that
uses an antibody against AdoHcy
and a fl uoro phore-conjugated
AdoHcy. The fl uorescent AdoHcy
conj ugate is bound by the antiAdoHcy-antibody to produce a high
FP complex: The AdoHcy produced
in the methy lation process displaces
the fluorescent tracer, resulting in
a decrease of the FP signal. Al
though the assay is homogenous
and continuous, its sensitivity is
low. Collazo et a/. 41 have reported an enzyme-coupled assay that utilizes 5-adenosylhomocysteine hy
drolase (SAHH) to hydrolyze the
methyltransferase product, AdoH
cy, to homocysteine (Hey) and
adenosine (Ado), and adenosine
deaminase to pull the reaction to
completion. The Hey concentration is then determined through conju
gation of its free sul fhyd ryl moiety
to a th iol-sensitive fluorophore
(Th ioGlo). One disadvantage of this
assay is its sensitivity to thiol-based
reducing agents (e.g., OTT or Pmercaptoethanol), including thiol
containing compounds. A disad
vantage of enzyme-coupled assays
© MARY ANN LIEBERT. INC. • VOL. XX NO. XX • XXXX 2013 ASSAY and Drug Development Technologies 7
HORIUCHI ET AL.
FIG. 4· The IC50 dete rminations of A120 8 120 control compounds for G9a under 100 100 different conditions. (A) G9a with o.5 11M of histone H3 (aa1-21) l:' 80 l:' 80 peptide as a s ubstrate at 10 11M > > u 60 u 60 SAM, and (B) G9a with 5 ~tM of < < histone H3 (aa1-21; K9-mono- .,. 40 .,. 40
methylated) peptide as substrate. 20 20 The obtained IC50 values are (A)
0 0 13.9 ~tM for SAH (e) and 2.2 ~tM for BIXo1294 ('i7) , (B) 2.4 and -9 -8 -7 -8 ·5 -4 -3 -9 -8 -7 -8 -5 -4 -3
1.3 IJM, respectively. Log [Compound] (M) Log [Compound] (M)
in general is the possibility of fa lse positives, wh ich are inhibitors of the coupling enzymes rather than the screening target, and the consequent need for counter assays fo r the coupling enzymes. In addition, the coupling enzyme should not be rate limiting to the overall reaction: SAini, however, is a very slow enzyme. A further
Table 4 . Mcthyltransferase Profiling for Suramin and Analogs
Target ! Substrate I Suramin I NFllO I NF449
OOTll Cort histone 2.12±0.50 - 6.54±0.91
EZH1 Core histone 45.9±20 100±31 36.2± 15
EZH2 Core histone 11.5± 10 116±15 13.6± 10
G9a Histone H3 29.5±5.2 198±22 39.4± 7.4
GlP Core histone 3.64±0.86 - 2.95±0.52
NSD2 Nucleosomes 0.32±0.04 - 1.66±0.42
SET18 Core histone 3.48± 1.02 - 1.92± 1.3
sm Histone H3 4.94± 1.14 - 11.6±2.4
SETS Histone H4 - 14.8±4.5 -SW39H1 Histone H3 54.6± 14 - 144± 42
SW39H2 Histone H3 30.3± 12 - -PRMT1 Histone H4 7.50±2.1 - 8.76± 2.4
PRMT3 Histone H3 10.4± 2.4 - 11.2±2.8
PRMT4 Histone H3 1.51±0.45 - 1.27±0.22
PRMT5 Histone H3 33.2± II - -PRMT6 Histone H3 3.32±0.12 - 3.35±0.03
SET MAR Histone H3 21.0± 7.2 - 23.9±2.8
SMYD2 Histone H3 1.18±0. 1 - 1.07±0.04
disadvantage of the SAHH/ThioGio system, which applies equally to any system based on fluorescence detection, is that fluorescence or fluo rescence quenching from screening compounds may interfere with the assay. The mobili ty-shi ft assay42 and the FLEXYTETW Fluorescence Lifetime assay have also been reported fo r G9a
I SAH
1.79±0.74
18.0±0.50
11.6±2.1
3.66±0.07
0.24±0.01
2.06±0.30
3.20±0.68
61.1±9.6
55.1±16
50.5± 11
45.6± 12
0.39±0.10
1.94±0.52
0.12±0.04
1.66±0.5
0.08±0.03
0.22±0.05
0.32±0.1
screen ing, requiring a protease to digest the unmethylated substrate for detection. These assays not only require specially designed artificial substrates, but also require counter screening for the coupling proteases. Further, a lack of hit overl ap with the FlashPlate assay has been reported.43 All of the disadvantages described above are minimized in the goldstandard miniaturized radioisotope-based HotSpot fo rmat. Since this format is based on the fil ter-binding capture of substrates, the only limitation is the capacity of the filter binding. However, the linear binding range is much larger than that of the FlashPiate. This can be overcome by diluting the reaction mixture before applying on the fil ter when the substrate concentration exceeds the binding capacity. The binding capacity and a linear range can be measured easily by a standard curve of known concentrations of substrates.
Profiles are reported as IC50o in j.tM. Boldface and italics indicate consistent and inconsistent inhibitions. respectively (see the text).
The 1C50 value of G9a inhibitor, BlXO 1294, was originally reported as 2.71JM for G9a with the DELFlA assay.33 Later, 1C50 values of 1.91JM, 180 nM, and 250 nM were reported with mass spectrometry [ 10 11M Histone HJ ( 1-15) at 100 11M SAM],34 enzyme-coupled assays [51JM HJ ( 1-25) at 161-lM SAM], and CLOT assays [0.5 1JM biotin-HJ (1- 11) at 20 11M SAM],35 respectively. Utilizing the HotSpot format [a standard condition of SIJM HJ ( 1-2 1) at 111M SAM), we obtained an IC50 value of 5.3 11M (Fig. J A). Considering the competitive mode of BIXO 1294 inhibition with respect to
SAH, 5-adenosyl-t-homocysteine.
8 ASSAY and Drug Development Technologies xxxx 2013
the peptide substrate and the K., for peptide (0.6 1JM in the Table 2), the K; value of
BIX01294 is estimated as 570 nM, in good agreement with published
data at low peptide conccntrations.35 Since it has been reponed that
BIXO 1294 is competitive with the peptide substrate, but uncompct
itive with SAM,33 the IC50 value was determined under lower peptide
and higher SAM concen trations. Under this condition [0.5 1JM 113 ( 1- 21) at 10 11M SAM], the IC50 value was 2.2 J.!M (Fig. 4A), shifted
about twofold, which is close to the value obtained by mass spcc
tromctry.3~ Interest ingly, the IC50 value was shifted about fourfold
lower when the s ubstrate was monomethylated (Figs. JA vs. 48).
However, the IC50 values a rc still higher than those compared to the
values obtained by an SAHH-coupling assay or CLOT assay.J5 In
assays using the Alpha LISA technique, the IC50 value of BIXO 1294
against G9a (2.21JM) was a lso higher (PerkinElmcr, AlphaLISA
Technical Note #2). We have performed another radioisotope-based
assay using strcptavidi n-eoatcd FlashPiate with biotinylated his
tone 113 peptide, and obtained similar results to those from the
HotSpot assay. When using histone H3 protein as substrate, the IC50
value was shifted 80-fold higher (Fig. JB). These sh ifts may be
caused by the change in binding affinity to substrates, since
BIX0 1294 is a competitive inhibitor with respect to the peptide
substra te.JJ As it is expected that the binding affin ity would in
crease for a protein substrate relative to a peptide, the increased IC50
value for a peptide/prote in competitive inhibitor would make sense,
consistent with a very low K, value for the protein substrate. In fact,
it was very hard to obtain the K, values for the protein substrate for
most HMTs. Shinkai and Tachibana44 have a lso observed tha t the
inhibi tion of G9a by BIXO 1294 is robust if an H3 N- tcrminal ol i
gopcptide is used as a substrate for the in vitro methyl transferase
assay, but is not significant (no inhibition at IO J.!M) if a fu ll - length
113 is used. It would be interesting to determine the processivity of
G9a mcthyltransfcrasc activity with a protein substra te in the
presence and absence of BIXO 1294. Further studies are needed to
elucidate the mechanism of action of BIXO 1294 (and recently found
analogs) not on ly with a peptide substrate, b ut with a protein sub
strate as well.
The data in this study demonstrate the capability of the HotSpot
platform when applied to the histone methyltransfcrasc assays. The
data quality is sufficient for all drug discovery activities, from ul tra
high throughput sc reening to compound profiling against a large
collection of HMTs and kinetic studies. Advantageous featu res of this
pla tform for drug discovery include the absence of interference from
nuoresccnt compounds and the e limination of the need for coupling
enzy mes, speci fic antibodies, or specifically modified peptide sub
strates. This enables substrate profiling as well as the determinatio n
of total methylation with unidentified protein substrates or with
known pcptides/protcins at undetermined methylation sites. Taking
these advantages, one can perform compound screening at K.,, o f
peptide o r SAM, or profiling under conditions close to in vivo using
nuclcosomcs as the substrate. One concern may be data reproduc
ibility when using nucleosomes or core histone as a substrate, since
they arc purified from natural sources (Hcla or chicken, respectively).
Although their methyla tion states are unknown, data reproducibility
was satisfactory (the data consistency of the IC50 values for SAil was
ASSAY DEVELOPMENT FOR HMTS
shown in Supplementary Table SJ); presumably, preparations are
well homogenized and minimal lot-to-lot variability. Since the
platform is a miniaturized radioisotope-based assay, it reduces the
cost by minimizing reagent usage. This is a considerable advantage especially for difficult or expensive enzymes and substrates. We
performed a small -library !ITS against DOT I L, which requires a
special substrate, core histones, and suramin was identified as a
DOTIL inhibitor. Subsequently, suramin was profiled against 17 methyltransferases with different substrates. Since the major ad
vantage of this assay format is that it can be applied universally to
methyltransfcrascs regardless of the substrate, it is suitable fo r pro
filing. Although the activities of some IIMTs arc increased at low
concentrations, mcthyltransferases that were consistently inhibited
(without apparent activation) by suramin a rc DOT I L, NSD2, and
PRMT4 with IC50 values at a low J.!M range (Table 4). This is the first
finding that suramin inhibits DOT I Land NSD2 activities, although it
has been reported very recently that a few IIMTs a rc inhibited by a
suramin analog using a peptide as the substrate.45 Suramin is an old
drug that has been used fo r the treatment of trypanosomiasis and is
known as an antagonist of P2 receptors; recently, the application of
suramin to cancer treatment has been explorcd.46 It would be in
teresting to determine the effects of suramin on methylatio n states at
the cellular level, especially in cancer cell lines.
ACKNOWLEDGMENT This wo rk was funded in part by NIH SBIR grants R44CA I 3962 1 to
II.M.
DISCLOSURE STATEMENT No competing financial interests exist.
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Address correspondence to: Kurumi Y. Horiuchi, PhD, or Haichi11g Ma, PhD
Departme111 of Biochemistry Reactio11 Biology Corporatio11
011e Great Valley Parkway, Suite 2
Malvern, PA 19355
E-mail: ku ru m [email protected]